U.S. patent number 6,965,062 [Application Number 10/247,857] was granted by the patent office on 2005-11-15 for tobacco cultivar nc 2000.
This patent grant is currently assigned to North Carolina State University. Invention is credited to Rebeca C. Rufty.
United States Patent |
6,965,062 |
Rufty |
November 15, 2005 |
**Please see images for:
( Certificate of Correction ) ** |
Tobacco cultivar NC 2000
Abstract
The present invention relates to a novel tobacco cultivar
designated NC 2000, which is resistant to blue mold caused by the
fungus Peronospora tabacina Adam. The invention provides seeds of
the cultivar NC 2000, plants and parts thereof of the cultivar NC
2000, a tissue culture derived from the cultivar NC 2000, hybrids
produced from cultivar NC 2000 and lines derived from cultivar NC
2000, as well as genetically modified forms of the foregoing plants
and tissue culture. Also provided are methods of producing cultivar
NC 2000 plants, cultivar NC 2000 hybrid plants, and tobacco lines
derived from cultivar NC 2000.
Inventors: |
Rufty; Rebeca C. (Raleigh,
NC) |
Assignee: |
North Carolina State University
(Raleigh, NC)
|
Family
ID: |
26938946 |
Appl.
No.: |
10/247,857 |
Filed: |
September 20, 2002 |
Current U.S.
Class: |
800/317.3;
800/260; 800/301; 435/414; 800/279; 800/303; 800/278; 800/300;
800/302; 800/265 |
Current CPC
Class: |
A01H
6/823 (20180501); A01H 5/12 (20130101) |
Current International
Class: |
A01H
5/12 (20060101); A01H 005/00 (); A01H 005/10 ();
A01H 001/00 (); C12N 015/82 (); C12N 005/04 () |
Field of
Search: |
;800/317,317.3,317.5,260,265,278,279,300,301,302,303 ;435/414 |
Other References
Cytogenetics of flower modification of a cytoplasmic male-sterile
tobacco D.U. Gerstel et al. 1980 Genetics 96:223-225. .
The effect of removing Leaf Surface Components with Acetone from
Immunized and Nonimmuzied Resistant Tobacco Plants on Their
Susceptibility to Blue Mold. S.Tuzun et al. 1989 The American
Phytopathological Society vol. 79, No 10 1024-1027. .
Brake et al.; "Use of Marker Assisted Selection to Screen for Blue
Mold Resistant in Burley Tobacco," Agronomy/Phytopatholoy Programme
for Accompanying Persons, Coresta, Lisbon, Portugal (Oct. 16-19,
2000). .
Milla; Thesis--"Identification of RAPD Markers Linked to Blue Mold
Resistance in Tobacco," NCSU--Department of Crop Science, Raleigh,
NC (1998). .
Rufty; "Genetics of Host Resistance to Tobacco Blue Mold," Chapter
5, Blue Mold of Tobacco (American Phytopathological Society Press)
141-164 (1989). .
Rufty et al., "Registration of NC-BMR 42 and NC-BMR 90 Germplasm
Lines of Tabacco," Crop Science 30:1 241-242 (1990). .
Wernsman et al.; "Tobacco" Chapter 17, Cultivar Development Crop
Species 669-698 (1987)..
|
Primary Examiner: Kubelik; Anne
Assistant Examiner: Para; Annette H
Attorney, Agent or Firm: Myers Bigel Sibley & Sajovec,
P.A.
Parent Case Text
RELATED APPLICATION INFORMATION
This application claims the benefit of U.S. Provisional Application
No. 60/323,908, filed Sep. 21, 2001, the disclosure of which is
incorporated by reference herein in its entirety.
Claims
That which is claimed is:
1. A tobacco seed designated NC 2000, representative seed of said
tobacco cultivar NC 2000 having been deposited under ATCC Accession
No. PTA-3721.
2. A tobacco plant, or thereof, produced by the seed of claim
1.
3. Pollen of the plant of claim 2.
4. An ovule of the plant of claim 2.
5. A tobacco plant, or a part thereof, having all the physiological
and morphological characteristics of tobacco cultivar NC 2000.
6. A tissue culture of regenerable cells of the plant of claim
2.
7. The tissue culture according to claim 6, the cells from a plant
part selected from the group consisting of leaves, pollen, embryos,
cotyledon, hypocotyl, roots, root tips, anthers, flowers and parts
thereof, ovules, shoots, stems, stalks, pith and capsules or
wherein the regenerable cells are callus or protoplasts derived
therefrom.
8. A tobacco plant regenerated from the tissue culture of claim 6,
wherein the plant expresses all the morphological and physiological
characteristics of tobacco cultivar NC 2000.
9. A tobacco plant having all of the physiological and
morphological characteristics of the tobacco plant of claim 2, said
tobacco plant having been produced by a tissue culture process
using the tobacco plant of claim 2 as the starting material.
10. A method for producing a first generation (F.sub.1) hybrid
tobacco seed wherein the method comprises crossing the plant of
claim 2 with a different inbred or doubled-haploid parent tobacco
plant and harvesting the resultant first generation (F.sub.1)
hybrid tobacco seed.
11. The method of claim 10, wherein the tobacco plant of claim 2 is
a female parent.
12. The method of claim 10, wherein the tobacco plant of claim 2 is
a male parent.
13. A method for producing a NC 2000-derived tobacco plant
expressing resistance to blue mold caused by the fungal pathogen
Peronospora tabacina Adam, wherein the method comprises: (a)
crossing tobacco cultivar NC 2000, representative seed of said
tobacco cultivar NC 2000 having been deposited under ATCC Accession
No. PTA-3721, with a second tobacco plant to yield progeny tobacco
seed; (b) growing said progeny tobacco seed, under plant growth
coditions, to yield said NC 2000-derived tobacco plant expressing
resistance to blue old caused by the fungal pathogen Peronospora
tabacina Adam.
14. An herbicide-resistant tobacco plant, or a part thereof,
produced by stably transforming the plant or part thereof of claim
8 with a transgene that confers herbicide resistance.
15. An herbicide-resistant tobacco plant, ro a part thereof,
produced by stably transforming the plant or part thereof of claim
5 with transgene that confers herbicide resistance.
16. A disease-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 8 with a
transgene that confers disease resistance.
17. An insect-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 8 with a
transgene that confers insect resistance.
18. A disease-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 5 with a
trangene that confers disease resistant.
19. An insect-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 5 with a
transgene that confers insect resistance.
20. An herbicide-resistant tobacco plant, or a part thereof,
produced by stably transforming the plant or part thereof of claim
2 with a transgene that confers herbicide resistance.
21. A disease-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 2 with a
transgene that confers disease resistance.
22. An insect-resistant tobacco plant, or a part thereof, produced
by stably transforming the plant or part thereof of claim 2 with a
transgene that confers insect resistance.
23. A method of making an herbicide-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 2 with
a transgene that confers herbicide resistance.
24. A method of making a disease-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 2 with
a transgene that confers disease resistance.
25. A method of making an insect-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 2 with
a transgene that confers insect resistance.
26. A method of making an herbicide-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 5 with
a transgene that confers herbicide resistance.
27. A method of making a disease-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 5 with
a transgene that confers disease resistance.
28. A method of making an insect-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 5 with
a transgene that onfers insect resistance.
29. A method of making an herbicide-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 8 with
a transgene that confers herbicide resistance.
30. A method of making a disease-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 8 with
a transgene that confers disease resistance.
31. A method of making an insect-resistant tobacco plant wherein
the method comprises stably transforming the plant of claim 8 with
a transgene that confers insect resistance.
32. A method of making a male sterile tobacco plant, wherein the
method comprises crossing the tobacco plant of any one of claims 2,
5, 8 or 9 with a tobacco plant that has cytoplasmic male
sterility.
33. The method of claim 10, wherein said different inbred or
doubled-haploid parent tobacco plant has cytoplasmic male
sterility.
34. The method of claim 13, wherein said second tobaco plant has
cytoplasmic male sterility.
Description
FIELD OF THE INVENTION
The present invention relates to tobacco breeding, in particular,
to a new tobacco cultivar designated NC 2000 that is resistant to
blue mold caused by the fungus Peronospora tabacina Adam.
BACKGROUND OF THE INVENTION
Tobacco (Nicotiana tabacum L.) is an important commercial crop in
the United States as well as in other countries. Blue mold is one
of the most significant foliar diseases of tobacco. When weather
conditions are favorable, the disease spreads rapidly and attacks
plants throughout the growing season. It can completely destroy
transplants in the bed. In the field, the presence of the pathogen
can be seen as brown necrotic spots on the leaves or as a systemic
infection.
Control of the pathogen can be achieved by two means: the use of
fungicides and the introduction of resistant varieties. The
development of fungicide resistant strains of the fungus has
increased the need for resistant varieties. Naturally occurring
host resistance to Peronospora tabacina exists among wild Nicotiana
species mainly of Australian origin, where the pathogen is endemic.
Transfer of resistance into cultivated tobacco from various sources
has been successfully achieved via interspecific hybridization. The
most widely used sources are N. debneyi accessions. Commercially
grown burley cultivars are either susceptible or very susceptible
to the disease, with the exception of TN 90, which is relatively
tolerant, but is not resistant.
Accordingly, it would be desirable to provide a tobacco cultivar
that demonstrates blue mold resistance.
There are numerous stages in the development of any novel,
desirable plant germplasm. Plant breeding begins with the analysis
and definition of problems and weaknesses of the current germplasm,
the establishment of program goals, and the definition of specific
breeding objectives. The next step is selection of germplasm that
possess the traits to meet the program goals. The aim is to combine
in a single variety an improved combination of desirable traits
from the parental germplasm. These important traits may include
higher yield, resistance to diseases and insects, better stems and
roots, tolerance to drought and heat, improved nutritional quality,
and better agronomic quality.
Choice of breeding or selection methods depends on the mode of
plant reproduction, the heritability of the trait(s) being
improved, and the type of cultivar used commercially (e.g., F.sub.1
hybrid cultivar, pureline cultivar, etc.). For highly heritable
traits, a choice of superior individual plants evaluated at a
single location may be effective, whereas for traits with low
heritability, selection should be based on mean values obtained
from replicated evaluations of families of related plants. Popular
selection methods commonly include pedigree selection, modified
pedigree selection, mass selection, and recurrent selection.
The complexity of inheritance influences the choice of breeding
method. Backcross breeding is used to transfer one or a few
favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes. The use of recurrent selection in self-pollinating
crops depends on the ease of pollination, the frequency of
successful hybrids from each pollination, and the number of hybrid
offspring from each successful cross.
Each breeding program should include a periodic, objective
evaluation of the efficiency of the breeding procedure. Evaluation
criteria vary depending on the goals and objectives, but should
include gain from selection per year based on comparisons to an
appropriate standard, overall value of the advanced breeding lines,
and number of successful cultivars produced per unit of input
(e.g., per year, per dollar expended, etc.).
Promising advanced breeding lines are typically tested and compared
to appropriate standards in environments representative of the
commercial target area(s) for three or more years. The best lines
are candidates for new commercial cultivars; those still deficient
in a few traits may be used as parents to produce new populations
for further selection.
An important task is the identification of individuals that are
genetically superior, because for most traits the true genotypic
value is masked by other confounding plant traits or environmental
factors. One method of identifying a superior plant is to observe
its performance relative to other experimental plants and to a
widely grown standard cultivar. If a single observation is
inconclusive, replicated observations provide a better estimate of
its genetic worth.
The goal of a tobacco breeding program is to develop new, unique
and superior tobacco cultivars and hybrids. The breeder typically
initially selects and crosses two or more parental lines, followed
by repeated selfing and selection, producing many new genetic
combinations. In tobacco, completely homozygous doubled-haploid
plants may also be generated (Burk et al., (1979) Science 206:585).
The breeder can theoretically generate billions of different
genetic combinations via crossing, selfing and mutations.
Each year, the plant breeder selects the germplasm to advance to
the next generation. This germplasm is grown under different
geographical, climate and soil conditions, and further selections
are then made, both during and at the end of the growing season.
The cultivars which are developed are unpredictable. This
unpredictability is because the breeder's selection occurs in
unique environments and there are millions of different possible
genetic combinations being generated. A breeder of ordinary skill
in the art cannot predict the final resulting lines, except in a
very general fashion. The same breeder cannot produce the same
cultivar twice by using the exact same original parents and the
same selection techniques. This unpredictability results in the
expenditure of large amounts of research monies to develop superior
new tobacco cultivars.
The development of new tobacco hybrids involves the development and
selection of tobacco breeding lines, the crossing of these breeding
lines and selection of superior hybrid crosses. The hybrid seed is
produced by manual crosses between selected male-fertile parents or
by using male sterility systems. Hybrid combinations are identified
and developed on the basis of certain single gene traits such as
leaf size or color, flower color, disease resistance or herbicide
resistance, and the like, which are expressed in a hybrid.
Additional data, such as yield and quality traits, on parental
lines as well as the phenotype of the hybrid influence the
breeder's decision to continue with the specific hybrid cross.
Pedigree breeding and recurrent selection breeding methods are used
to develop true breeding cultivars from breeding populations.
Breeding programs combine desirable traits from two or more
cultivars or various broad-based sources into breeding pools from
which cultivars are developed by selfing or alternatively, by
creating doubled-haploids, and selection of desired phenotypes. The
new cultivars are evaluated to determine which have commercial
potential.
Pedigree breeding is commonly used for the improvement of
self-pollinating crops and parental lines for hybrids. Two parents
which possess favorable, complementary traits are crossed to
produce an F.sub.1. An F.sub.2 population is produced by selfing
one or several F.sub.1 plants. Selection of the best individuals
may begin in the F.sub.2 population; then, beginning in the
F.sub.3, the best individuals in the families are selected.
Replicated testing of families can begin in the F.sub.4 generation
to improve the effectiveness of selection for traits with low
heritability. At an advanced stage of inbreeding (i.e., F.sub.6 and
F.sub.7), the best lines or mixtures of phenotypically similar
lines are tested for potential release as new cultivars.
Mass and recurrent selections can be used to improve populations of
either self- or cross-pollinating crops. A genetically variable
population of heterozygous individuals is either identified or
created by intercrossing several different parents. The best plants
are selected based on individual superiority, outstanding progeny,
or excellent combining ability. The selected plants are
intercrossed to produce a new population in which further cycles of
selection are continued.
Backcross breeding has been used to transfer genes for a simply
inherited, highly heritable trait into a desirable homozygous
cultivar or inbred line which is the recurrent parent. The source
of the trait to be transferred is called the donor parent. The
resulting plant is expected to have the attributes of the recurrent
parent (e.g., cultivar) and the desirable trait transferred from
the donor parent. After the initial cross, individuals possessing
the phenotype of the donor parent are selected and repeatedly
crossed (backcrossed) to the recurrent parent. The resulting plant
is expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent.
The single-seed descent procedure in the strict sense refers to
planting a segregating population, harvesting a sample of one seed
per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F.sub.2
to the desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2, individuals. The
number of plants in a population declines in each generation due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
In a multiple-seed procedure, tobacco breeders harvest seeds from
one or more flowers from each plant in a population and pool them
to form a bulk. Part of the bulk is used to plant the next
generation and part is put in reserve. The procedure has been
referred to as modified single-seed descent technique.
Proper testing should detect any major faults and establish the
level of superiority or improvement over current cultivars. In
addition to showing superior performance, there must be a demand
for a new cultivar that is compatible with industry standards or
which creates a new market. The introduction of a new cultivar will
incur additional costs to the seed producer, the grower, the
processor and the consumer, for special advertising and marketing,
altered seed and commercial production practices, and new product
utilization. The testing preceding release of a new cultivar should
take into consideration research and development costs as well as
technical superiority of the final cultivar. For seed-propagated
cultivars, it must be feasible to produce seed easily and
economically.
Maternal haploids can be obtained by pollination of plants of N.
tabacum with N. africana. Numerous seeds develop in fruits from
this cross, but germinating interspecific hybrid seedlings are
highly lethal (99.9%). Surviving F.sub.1 plants consist of mixtures
of aneuploid interspecific hybrids and maternal haploids. The
chromosomes of the maternal haploids are derived from the N.
tabacum female plant. The procedure is very simple, but requires
technical skill to distinguish phenotypically the aneuploid
interspecific hybrids from maternal haploids in seedling stages.
Environmental effects on tobacco females crossed with N. africana
pollen greatly influence the number of haploids obtained per
capsule. One to three haploid plants frequently can be obtained
from a capsule of a tobacco.times. N. africana cross when the
tobacco female is grown in the field. Haploid numbers per
pollination of greenhouse-grown tobacco are five to ten times
lower. Chromosome-doubled haploids obtained by this technique are
superior to ADH lines from the same parental sources and more
closely resemble the performance of conventionally developed inbred
genotypes.
Methods of tobacco breeding are discussed in detail in Wernsman, E.
A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages
669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.),
MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.
SUMMARY OF THE INVENTION
The present invention relates to a new and distinctive
doubled-haploid tobacco cultivar designated NC 2000, which is the
result of years of careful breeding and selection, and is highly
resistant to blue mold. As far as the inventor is aware, NC 2000 is
the first blue mold resistant burley cultivar.
The invention further provides seeds of the cultivar NC 2000,
plants of the cultivar NC 2000, tissue culture comprising tissue,
callus, cells or protoplasts of the cultivar NC 2000, hybrids
having a cultivar NC 2000 parent or ancestor, and NC 2000 derived
tobacco plants, as well as genetically modified (e.g., by
conventional breeding or genetic engineering techniques) forms of
the foregoing plants and tissue culture. The present invention
further provides methods of producing a tobacco plant by crossing
the NC 2000 cultivar with itself or a different tobacco line. The
invention further relates to methods for producing other tobacco
cultivars or breeding lines derived from cultivar NC 2000.
These and other aspects of the invention are set forth in more
detail in the description of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 and 2. RAPD reaction of individual tobacco varieties
(controls) and doubled haploid lines.
FIG. 3. The fifty doubled haploid lines showing % LAD found in
field evaluations and resistant and susceptible classifications by
use of markers. Higher reliability of the markers is found at the
extremes of a tobacco plant's resistance or susceptibility to blue
mold.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
The terminology used in the description of the invention herein is
for the purpose of describing particular embodiments only and is
not intended to be limiting of the invention. As used in the
description of the invention and the appended claims, the singular
forms "a", "an" and "the" are intended to include the plural forms
as well, unless the context clearly indicates otherwise.
As used herein, a tobacco plant that is "resistant" to blue mold or
blue mold "resistance" is intended to indicate that the plant has a
significantly reduced size and/or incidence of lesions induced by
the pathogen Peronospora tabacina Adam as compared with a control
susceptible variety (e.g., KY 14 or the like) under conditions of
infestation. Resistance may be evaluated by any suitable method
known in the art, e.g., by determining the percentage leaf area
damage induced by blue mold. The term "resistant/resistance" is not
intended to indicate that the plant is absolutely immune from blue
mold. Those skilled in the art will appreciate that the degree of
resistance may be assessed with respect to a plurality or even an
entire field of plants. A tobacco strain may be considered
"resistant" to blue mold if the overall incidence and/or size of
lesions is reduced, even if particular, individual, plants may be
susceptible to disease.
In embodiments of the invention, the blue mold resistant plants of
the invention have one or more (e.g., two or more, three or more,
four and more, etc.) of the coupling markers listed in Table 5.
Likewise, in embodiments of the invention, the blue mold resistant
tobacco plants lack one or more of the repulsion markers listed in
Table 5. In particular embodiments, the tobacco plants of the
invention have all of the coupling markers in Table 5 and/or lack
all of the repulsion markers of Table 5.
Description of the Variety.
Burley Tobacco Cultivar NC 2000, tested and developed as DH 408, is
a doubled-haploid line derived from the cross of NC BMR-113 (a blue
mold resistant germplasm line released by the North Carolina
Agricultural Research Service in 1992) X KY 14. Doubled-haploid
lines were obtained from F.sub.1 hybrids of this cross using the N.
africana method for generating maternal haploids (Burk et al.,
(1979) Science 206:585) followed by chromosome doubling using the
in vitro mid-vein culture technique (Kasperbauer and Collins,
(1972) Crop. Sci. 12:98).
Several hundred doubled-haploid lines (F.sub.1 -derived) were
grown. The plants were bagged to prevent cross-pollination and the
seeds collected. Five plants from each doubled-haploid line were
grown, the plants bagged, and the seeds collected and pooled for
each line for two consecutive years prior to field trials.
In 1992 through 1996, the doubled-haploid lines were field tested
for blue mold resistance in Papantla, Veracruz, Mexico, where blue
mold is endemic. As a result of the field test results, line DH 408
was selected for its stable, uniform and high-level of resistance
to blue mold, and was eventually re-designated as NC 2000. NC 2000
was further characterized in field studies at the Mountain Research
Station (Waynesville, N.C.) and the Upper Mountain Research Station
(Laurel Springs, N.C.) and in the 1998 Regional Burley Variety
Evaluation Test.
NC 2000 is highly resistant to blue mold caused by the fungus
Peronospora tabacina Adam. Percent leaf area damage ratings of NC
2000 to blue mold are significantly lower than disease values of
any commercial burley cultivar evaluated for this trait. Because
the NC 2000 cultivar is not completely immune to blue mold, a
minimum number of fungicide applications may be necessary during
prolonged cool and wet periods, which are highly conducive to blue
mold development.
NC 2000 has all of the coupling markers and lacks all of the
repulsion markers shown in Table 5.
NC 2000 is also resistant to tobacco mosaic virus (TMV) and
wildfire (Pseudomonas syringae pv. tabaci), but susceptible to
black shank (races 1 and 0), black root rot, and the polyvirus
complex.
Yielding ability of NC 2000 compares well with commercial cultivars
and has a weighted grade index equivalent to VA 509 and modestly
higher than KY 14. NC 2000 has acceptable levels of nicotine and
total alkaloids. Results of the 1998 Regional Burley Evaluation
Test indicate that NC 2000 meets minimum quality standards and
smoke flavor is acceptable.
NC 2000 is a pure doubled-haploid line selected from a single plant
and, therefore, is completely homozygous. The resistance of NC 2000
to tobacco blue mold caused by the fungus Peronospora tabacina Adam
has remained stable and uniform within commercially acceptable
limits over at least eight generations. No variants in blue mold
resistance have been observed to date.
Additional morphological and physiological characterization of
cultivar NC 2000 is found in Appendix A, which is attached hereto.
Although NC 2000 is a pure line derived from a single
doubled-haploid plant, NC 2000 shows about 5% of off-type plants,
primarily attributable to variations in the leaf shape and leaf
angle.
Other Embodiments of the Invention
The present invention also encompasses hybrid plants produced from
tobacco cultivar NC 2000, tobacco plants derived from NC 2000, and
NC 2000 plants comprising a gene that has been introduced therein
by traditional breeding or genetic engineering techniques, and
seeds, plant parts, and tissue cultures of the foregoing plants, as
well as methods of producing the plants of the invention.
I. Male Sterile Plants.
Tobacco can be bred by both self-pollination and cross-pollination
techniques. Individual tobacco flowers have both male and female
reproductive organs, and tobacco is naturally self-pollinating. It
is known in the art that it is often advantageous to create male
sterile/female fertile plants so that self-pollination can be
controlled.
Male sterile tobacco plants may be produced by any method known in
the art. Methods of producing male sterile tobacco are described in
Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco.
Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr
(ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.
A reliable method of controlling male fertility in plants offers
the opportunity for improved plant breeding. This is especially
true for development of tobacco hybrids, which typically relies
upon some sort of male sterility system. There are several options
for controlling male fertility available to breeders, such as:
manual or mechanical emasculation, cytoplasmic male sterility,
genetic male sterility, gametocides and the like. In one approach,
alternate strips of two tobacco lines are planted in a field, and
the male portions of flowers are removed from one of the lines
(female). Providing that there is sufficient isolation from sources
of foreign tobacco pollen, the emasculated plant will be fertilized
only from the other line (male), and the resulting seed is
therefore hybrid and will form hybrid plants.
The laborious, and occasionally unreliable, mechanical emasculation
process can be avoided by using cytoplasmic male-sterile (CMS)
lines. Plants of a CMS line are male sterile as a result of factors
resulting from the cytoplasmic, as opposed to the nuclear, genome.
Thus, this characteristic is inherited exclusively through the
female parent in tobacco plants, since only the female provides
cytoplasm to the fertilized seed. CMS plants are fertilized with
pollen from another line that is not male-sterile. Pollen from the
second line may or may not contribute genes that make the hybrid
plants male-fertile.
Alternative approaches of conferring genetic male sterility are
also suitable, such as multiple mutant genes at separate locations
within the genome that confer male sterility and chromosomal
translocations.
Still further methods of conferring genetic male sterility use a
variety of approaches such as delivering into the plant a gene
encoding a cytotoxic substance associated with a male tissue
specific promoter or an antisense system in which a gene critical
to male fertility is identified and an antisense to that gene is
inserted in the plant.
Another system useful in controlling male fertility makes use of
gametocides. Gametocides do not involve a genetic system, but
rather a topical application of chemicals. These chemicals affect
cells that are critical to male fertility. The application of these
chemicals affects fertility in the plants only for the growing
season in which the gametocide is applied (see U.S. Pat. No.
4,936,904). Application of the gametocide, timing of the
application and genotype specificity often limit the usefulness of
the approach.
II. Hybrid Production.
The use of male sterile lines is but one factor in the production
of tobacco hybrids. The development of tobacco hybrids involves, in
general, the development of completely homozygous lines, the
crossing of these lines, and the evaluation of the crosses. In the
case of tobacco, a completely homozygous line may be an inbred or a
doubled-haploid line.
Pedigree breeding and recurrent selection breeding methods are
typically used to develop inbred lines from breeding populations.
Breeding programs combine the genetic backgrounds from two or more
inbred lines or various other germplasm sources into breeding pools
from which new inbred lines are developed by selfing and selection
of desired phenotypes. The new inbreds are crossed with other
inbred lines or doubled-haploid lines, and the hybrids from these
crosses are evaluated to determine which of those have commercial
potential.
Doubled-haploid breeding is a more rapid method for producing
completely homozygous tobacco plants (Burk et al., (1979) Science
206:585). Haploid plants or cultures of haploid cells or tissues
are produced and chromosome doubling is induced, for example, by
colchicine treatment or by the midvein culture technique.
Doubled-haploid plants are regenerated following chromosomal
doubling.
Pedigree breeding starts with the crossing of two genotypes, each
of which may have one or more desirable characteristics that is
lacking in the other or which complements the other. If the two
original parents do not provide all the desired characteristics,
other sources can be included in the breeding population. In the
pedigree method, superior plants are selfed and selected in
successive generations. In the succeeding generations, the
heterozygous condition gives way to homogeneous lines as a result
of self-pollination and selection. Typically in the pedigree method
of breeding, five or more generations of selfing and selection is
practiced.
A single cross tobacco hybrid results from the cross of two inbred
or doubled-haploid lines, or from the cross of an inbred with a
doubled-haploid line, each of the parents having a genotype that
complements the genotype of the other. The hybrid progeny of the
first generation is designated F.sub.1. Preferred F1 hybrids may be
more vigorous than their inbred parents. This hybrid vigor, or
heterosis, can be manifested in many polygenic traits, including
increased vegetative growth and increased yield.
In general, the development of a tobacco hybrid involves three
steps: (1) the selection of plants from various germplasm pools for
initial breeding crosses; (2) the selfing of the selected plants
from the breeding crosses for several generations to produce a
series of inbred lines, which, although different from each other,
breed true and are highly uniform and/or the production of a series
of doubled-haploid lines; and (3) crossing the selected inbred
and/or doubled-haploid lines with different inbred/doubled-haploid
lines to produce the hybrid progeny (F.sub.1). A consequence of the
homozygosity and homogeneity of the inbred and/or doubled-haploid
lines is that the hybrid between a defined pair of
inbreds/doubled-haploids will always be the same. Once the parents
that give a superior hybrid have been identified, the hybrid seed
can be reproduced indefinitely as long as the homogeneity of the
parents is maintained.
A single cross hybrid is produced when two lines are crossed to
produce the F.sub.1 progeny. A double cross hybrid is produced from
four inbred and/or doubled-haploid lines crossed in pairs
(A.times.B and C.times.D) and then the two F.sub.1 hybrids are
crossed again (A.times.B).times.(C.times.D). Much of the hybrid
vigor exhibited by F.sub.1 hybrids is generally lost in the next
generation (F.sub.2). Consequently, seed from hybrids is not
typically used for planting stock.
As described above, hybrid seed production regimes generally use
male sterile/female fertile parent plants. Incomplete removal or
inactivation of the pollen provides the potential for self
pollination. This inadvertently self pollinated seed may be
unintentionally harvested and packaged with hybrid seed. Once the
seed is planted, it is possible to identify and select these self
pollinated plants due to their decreased vigor. These
self-pollinated plants will be genetically equivalent to the female
inbred line used to produce the hybrid. Female selfs are identified
by their less vigorous, appearance for vegetative and/or
reproductive characteristics as is known in the art.
Identification of these self-pollinated lines can also be
accomplished through molecular marker analyses. Through these
technologies, the homozygosity of the self-pollinated line can be
verified by analyzing allelic composition at various loci along the
genome.
III. Evaluation of Plants for Homozygosity and Phenotypic
Stability.
It is desirable and advantageous for a tobacco cultivar to be
highly homogeneous, homozygous and phenotypically uniform and
stable for use as a commercial cultivar. In the case of
double-haploids, these plants are generated so as to be completely
homozygous and uniform. In the case of inbreds or other lines,
there are many analytical methods available to determine the
homozygotic and phenotypic stability of the variety.
The oldest and most traditional method of analysis is the
observation of phenotypic traits. The data is usually collected in
field experiments over the life of the tobacco plants to be
examined. Phenotypic characteristics most often observed are for
traits associated with seed yield, disease resistance, maturity,
plant height, flower color, leaf color, leaf yield, leaf size, leaf
angle, and concentration of chemical components such as nicotine,
total alkaloids or reducing sugars.
In addition to phenotypic observations, the genotype of a plant can
also be examined. There are many laboratory-based techniques
available for the analysis, comparison and characterization of
plant genotypes; among these are Isozyme Electrophoresis,
Restriction Fragment Length Polymorphisms (RFLPs), Randomly
Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase
Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF),
Sequence Characterized Amplified Regions (SCARs), Amplified
Fragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats
(SSRs) which are also referred to as Microsatellites.
As described in Examples 2 and 3, the tobacco BMR locus, which
confers resistance to blue mold, has been found to be linked to 21
markers (Table 5). Some of these markers (UBC-149, UBC-180,
UBC-534, UBC-544, UBC-610, UBC-240) are particularly reliable for
determining whether a plant is resistant to blue mold. As described
above, in embodiments of the invention, the claimed tobacco plant
has one or more of the coupling markers and/or lacks one or more of
the repulsion markers shown in Table 5.
The presence or absence of the marker in the plant genotype may be
determined by any method known in the art. For example, the marker
sequence (or its complement) may be used as a hybridization probe,
e.g., for Southern or in situ analysis of genomic DNA. Typically,
however, due to greater ease and sensitivity, an amplification
method, such as PCR will be used to detect the presence or absence
of the marker in the plant genotype.
The molecular markers disclosed herein can be used in any method of
nucleic acid amplification known in the art. Such methods include
but are not limited to Polymerase Chain Reaction (PCR; described in
U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188), Strand
Displacement Amplification (SDA; described by G. Walker et al.,
Proc. Nat Acad. Sci. USA 89, 392 (1992); G. Walker et al., Nucl.
Acids Res. 20, 1691 (1992); U.S. Pat. No. 5,270,184), thermophilic
Strand Displacement Amplification (tSDA; EP 0 684 315 to Frasier et
al.), Self-Sustained Sequence Replication (3SR; J. C. Guatelli et
al., Proc Natl. Acad. Sci. USA 87,1874-78 (1990)), Nucleic Acid
Sequence-Based Amplification (NASBA; U.S. Pat. No. 5,130,238 to
Cangene), the Q.beta. replicase system (P. Lizardi et al.,
BioTechnology 6, 1197 (1988)), or transcription based amplification
(D. Y. Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173-77
(1989)).
IV. Transfer of Traits into Tobacco Cultivar NC 2000.
Genetic variants of NC 2000 that are naturally-occurring or created
through traditional breeding methods using cultivar NC 2000 are
also intended to be within the scope of this invention. In
particular embodiments, the invention encompasses plants of
cultivar NC 2000 and parts thereof further comprising one or more
additional traits, in particular, specific, single gene transferred
traits. Examples of traits that may be transferred include, but are
not limited to, herbicide resistance, disease resistance (e.g.,
bacterial fungal or viral disease), nematode resistance, yield
enhancement, improved nutritional quality (e.g., oil starch and
protein content or quality), altered chemical composition (e.g.,
nicotine, total alkaloids, reducing sugars), improved leaf
characteristics (color, shape, size, number, angle), or other
agronomically important traits.
Such traits may be introgressed into cultivar NC 2000 from another
tobacco cultivar or may be directly transformed into cultivar NC
2000 (discussed below). Preferably, one or more new traits are
transferred to cultivar NC 2000, or, alternatively, one or more
traits of cultivar NC 2000 are altered or substituted. The
introgression of the trait(s) into cultivar NC 2000 may be achieved
by any method of plant breeding known in the art, for example,
pedigree breeding, backcrossing, doubled-haploid breeding, and the
like (see, Wernsman, E. A., and Rufty, R. C. 1987. Chapter
Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop
Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New
York, N.Y. 761 pp.).
The laboratory-based techniques described above, in particular RFLP
and SSR, can be used in such backcrosses to identify the progenies
having the highest degree of genetic identity with the recurrent
parent. This permits one to accelerate the production of tobacco
cultivars having at least 90%, preferably at least 95%, more
preferably at least 99% genetic identity with the recurrent parent,
yet more preferably genetically identical to the recurrent parent,
and further comprising the trait(s) introgressed from the donor
patent. Such determination of genetic identity can be based on
molecular markers used in the laboratory-based techniques described
above.
The last backcross generation is then selfed to give pure breeding
progeny for the gene(s) being transferred. The resulting plants
generally have essentially all of the morphological and
physiological characteristics of cultivar NC 2000, in addition to
the trait(s) (e.g., one or more single gene traits) transferred to
the inbred. The exact backcrossing protocol will depend on the
trait being altered to determine an appropriate testing protocol.
Although backcrossing methods are simplified when the trait being
transferred is a dominant allele, a recessive allele may also be
transferred. In this instance, it may be necessary to introduce a
test of the progeny to determine if the desired trait has been
successfully transferred.
Those skilled in the art will appreciate that the tobacco genes
described below in connection with tobacco plants produced by
genetic engineering techniques may also be introduced into cultivar
NC 2000 by conventional breeding methods.
V. Transformation of Tobacco.
With the advent of molecular biological techniques that have
allowed the isolation and characterization of genes that encode
specific protein products, scientists in the field of plant biology
developed a strong interest in engineering the genome of plants to
contain and express foreign genes, or additional, or modified
versions of native or endogenous genes (perhaps driven by different
promoters) in order to alter the traits of a plant in a specific
manner. Such foreign, additional and/or modified genes are referred
to herein collectively as "transgenes." The term "transgene," as
used herein, is not necessarily intended to indicate that the
foreign gene is from a different plant species. For example, the
transgene may be a particular allele derived from another tobacco
line or may be an additional copy of an endogenous gene. Over the
last fifteen to twenty years several methods for producing
transgenic plants have been developed. Therefore, in particular
embodiments, the present invention also encompasses transformed
versions of the tobacco cultivar NC 2000.
Plant transformation involves the construction of an expression
vector that will function in plant cells. Such a vector comprises
DNA or RNA comprising a gene under control of, or operatively
linked to, a regulatory element (for example, a promoter). The
expression vector may contain one or more such operably linked
gene/regulatory element combinations. The vector(s) may be in the
form, e.g., of a plasmid or a virus, and can be used, alone or in
combination with other vectors, to provide transformed tobacco
plants, using transformation methods as described below to
incorporate transgenes into the genetic material of the tobacco
plant(s).
Any transgene(s) known in the art may be introduced into a tobacco
plant, tissue, cell or protoplast according to the present
invention, e.g., to improve commercial or agronomic traits,
herbicide resistance, disease resistance (e.g., to a bacterial
fungal or viral disease), nematode resistance, yield enhancement,
nutritional quality (e.g., oil starch and protein content or
quality), leaf characteristics (color, shape, size, number, angle),
and altered reproductive capability (e.g., male sterility) or
chemical composition (e.g., nicotine, total alkaloids, reducing
sugars). Alternatively, a transgene may be introduced for the
production of recombinant proteins (e.g., enzymes) or
metabolites.
In particular embodiments of the invention a transgene conferring
glyphosate resistance is introduced into the tobacco plant.
Alternatively, a transgene conferring disease resistance is
introduced. Exemplary transgenes are those conferring resistance to
Tobacco Mosaic Virus, Tobacco etch virus, Tobacco vein mottling
virus, Black root rot, Potato Virus Y, Bacterial wilt (Pseudomonas
solanacearum), Black shank fungus (Phythophthora parasitica), wild
fire (Pseudomonas syringae), and root knot nematodes.
In other embodiments, the transgene encodes an antisense RNA or any
other non-translated RNA as is known in the art.
A. Expression Vectors for Tobacco Transformation.
1. Marker Genes.
Expression vectors typically include at least one genetic marker,
operably linked to a regulatory element (a promoter, for example)
that allows transformed cells containing the marker to be either
recovered by negative selection, i.e., inhibiting growth of cells
that do not contain the selectable marker gene, or by positive
selection, i.e., screening for the product encoded by the genetic
marker. Many commonly used selectable marker genes for plant
transformation are well known in the transformation art, and
include, for example, genes that code for enzymes that
metabolically detoxify a selective chemical agent which may be an
antibiotic or a herbicide, or genes that encode an altered target
which is insensitive to the inhibitor. A few positive selection
methods are also known in the art.
One commonly used selectable marker gene for plant transformation
is the neomycin phosphotransferase II (npfII) gene, isolated from
transposon Tn5, which when placed under the control of plant
regulatory signals confers resistance to kanamycin (Fraley et al.,
(1983) Proc. Natl. Acad. Sci. U.S.A., 80: 4803). Another commonly
used selectable marker gene is the hygromycin phosphotransferase
gene, which confers resistance to the antibiotic hygromycin (Vanden
Elzen et al., (1985) Plant Mol. Biol., 5: 299).
Additional selectable marker genes of bacterial origin that confer
resistance to antibiotics include gentamycin acetyl transferase,
streptomycin phosphotransferase, aminoglycoside-3'-adenyl
transferase, the bleomycin resistance determinant (Hayford et al.,
(1988) Plant Physiol. 86: 1216; Jones et al., (1987) Mol. Gen.
Genet., 210: 86; Svab et al., (1990) Plant Mol. Biol. 14: 197;
Hille et al., (1986) Plant Mol. Biol. 7: 171). Other selectable
marker genes confer resistance to herbicides such as glyphosate,
glufosinate or bromoxynil (Comai et al., (1985) Nature 317: 741;
Gordon-Kamm et al., (1990) Plant Cell 2: 603; and Stalker et al.,
(1988) Science 242: 419).
Other selectable marker genes for plant transformation are not of
bacterial origin. These genes include, for example, mouse
dihydrofolate reductase, plant 5eno/pyruvylshikimate-3-phosphate
synthase and plant acetolactate synthase (Eichholtz et al., (1987)
Somatic Cell Mol. Genet. 13: 67; Shah et al., (1986) Science 233:
478; Charest et al., (1990) Plant Cell Rep. 8: 643).
Another class of marker genes for plant transformation requires
screening of presumptively transformed plant cells rather than
direct genetic selection of transformed cells for resistance to a
toxic substance such as an antibiotic. These genes are particularly
useful to quantify or visualize the spatial pattern of expression
of a gene in specific tissues and are frequently referred to as
reporter genes because they can be fused to a gene or gene
regulatory sequence for the investigation of gene expression.
Commonly used genes for screening presumptively transformed cells
include .beta.-glucuronidase (GUS), .beta.-galactosidase,
luciferase and chloramphenicol acetyltransferase (Jefferson, R. A.,
(1987) Plant Mol. Biol. Rep. 5: 387; Teeri et al., (1989) EMBO J.
8: 343; Koncz et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84:131;
De Block et al., (1984) EMBO J. 3: 1681).
In vivo methods for visualizing GUS activity that do not require
destruction of plant tissue are also available (Molecular Probes
Publication 2908, Imagene Green.TM., p. 1-4 (1993) and Naleway et
al., (1991) J. Cell Biol. 115: 15). However, these in vivo methods
for visualizing GUS activity have not proven useful for recovery of
transformed cells because of low sensitivity, high fluorescent
backgrounds, and limitations associated with the use of luciferase
genes as selectable markers.
In addition, a gene encoding Green Fluorescent Protein (GFP) has
been utilized as a marker for gene expression in prokaryotic and
eukaryotic cells (Chalfie et al., (1994) Science 263: 802). GFP and
mutants of GFP may be used as screenable markers.
2. Promoters.
Genes included in expression vectors are typically driven by a
nucleotide sequence comprising a regulatory element, for example, a
promoter. Several types of promoters are now well known in the
transformation art, as are other regulatory elements that can be
used alone or in combination with promoters.
As used herein "promoter" includes reference to a region of DNA (or
RNA) upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific". A "cell type" specific promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter is a promoter which is under environmental
control. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions
or the presence of light. Tissue-specific, tissue-preferred, cell
type specific, and inducible promoters are included in the class of
"non-constitutive" promoters. A "constitutive" promoter is a
promoter which is active under most environmental conditions.
(A) Inducible Promoters.
An inducible promoter may be operably linked to a gene for
expression in tobacco. Optionally, the inducible promoter is
operably linked to a nucleotide sequence encoding a signal sequence
which is operably linked to a gene for expression in tobacco. With
an inducible promoter the rate of transcription increases in
response to an inducing agent.
Any inducible promoter can be used in the instant invention (see,
Ward et al., (1993) Plant Mol. Biol. 22: 361). Exemplary inducible
promoters include, but are not limited to, that from the ACEI
system which responds to copper (Melt et al., (1993) PNAS 90:
4567); the In2 gene from maize which responds to benzenesulfonamide
herbicide safeners (Hershey et al., (1991) Mol. Gen. Genetics 227:
229 and Gatz et al., (1994) Mol. Gen. Genetics 243: 32) or the Tet
repressor from Tn10 (Gatz et al., (1991) Mol. Gen. Genet. 227:
229). A particularly preferred inducible promoter is a promoter
that responds to an inducing agent to which plants do not normally
respond. An exemplary inducible promoter is the inducible promoter
from a steroid hormone gene, the transcriptional activity of which
is induced by a glucocorticosteroid hormone (Schena et al., (1991)
Proc. Natl. Acad. Sci. U.S.A. 88: 421).
(B) Constitutive Promoters.
In other embodiments, a constitutive promoter is operably linked to
a gene for expression in tobacco or the constitutive promoter is
operably linked to a nucleotide sequence encoding a signal sequence
which is operably linked to a gene for expression in tobacco.
Many different constitutive promoters can be utilized in the
instant invention. Exemplary constitutive promoters include, but
are not limited to, the promoters from plant viruses such as the
35S promoter from CaMV (Odell et al., (1985) Nature 313: 810) and
the promoters from such genes as rice actin (McElroy et al., (1990)
Plant Cell 2: 163); ubiquitin (Christensen et al., (1989) Plant
Mol. Biol 12: 619 and Christensen et al., (1992) Plant Mol. Biol.
18: 675); pEMU (Last et al., (1991) Theor. Appl. Genet. 81: 581);
MAS (Velten et al., (1984) EMBO J. 3: 2723) and maize H3 histone
(Lepelit et al., (1992) Mol. Gen. Genet. 231: 276 and Atanassova et
al., (1992) Plant Journal 2: 291).
The ALS promoter, a XbaI/NcoI fragment 5' to the Brassica napus
ALS3 structural gene (or a nucleotide sequence that has substantial
sequence similarity to said XbaI/NcoI fragment), represents a
particularly useful constitutive promoter (see, PCT publication WO
96/30530).
(C) Tissue-Specific or Tissue-Preferred Promoters.
In still other embodiments, a tissue-specific promoter is operably
linked to a gene for expression in tobacco. Optionally, the
tissue-specific promoter is operably linked to a nucleotide
sequence encoding a signal sequence which is operably linked to a
gene for expression in tobacco. Plants transformed with a gene of
interest operably linked to a tissue-specific promoter produce the
protein product of the transgene exclusively, or preferentially, in
a specific tissue.
Any tissue-specific or tissue-preferred promoter can be utilized in
the instant invention. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to, a
root-preferred promoter, such as that from the phaseolin gene
(Murai et al., (1983) Science 23: 476 and Sengupta-Gopalan et al.,
(1985) Proc. Natl. Acad. Sci. USA 82: 3320); a leaf-specific and
light-induced promoter such as that from cab or rubisco (Simpson et
al., (1985) EMBO J. 4: 2723 and Timko et al., (1985) Nature 318:
579); an anther-specific promoter such as that from LAT52 (Twell et
al., (1989) Mol. Gen. Genet. 217: 240); a pollen-specific promoter
such as that from Zm13 (Guerrero et al., (1993) Mol. Gen. Genet
224: 161) or a microspore-preferred promoter such as that from apg
(Twell et al., (1993) Sex. Plant Reprod. 6: 217).
3. Signal Sequences for Targeting Proteins to Subcellular
Compartments.
Transport of proteins produced by transgenes to a subcellular
compartment such as the chloroplast, vacuole, peroxisome,
glyoxysome, cell wall or mitochondrion, or for secretion into the
apoplast, may be accomplished by means of operably linking the
nucleotide sequence encoding a signal sequence typically at the 5'
and/or 3' region of a gene encoding the protein of interest.
Association of targeting sequences with the structural gene may
determine, during protein synthesis and processing, where the
encoded protein is ultimately compartmentalized. The presence of a
signal sequence directs a polypeptide to either an intracellular
organelle or subcellular compartment or for secretion to the
apoplast. Many signal sequences are known in the art (see, for
example, Becker et al., (1992) Plant Mol. Biol. 20: 49; Close, P.
S., Master's Thesis, Iowa State University (1993); Knox, C., et
al., (1987) Plant Mol. Biol. 9: 3; Lerner et al., (1989) Plant
Physiol. 91: 124; Fontes et al., (1991) Plant Cell 3: 483; Matsuoka
et al., (1991) Proc. Natl. Acad. Sci. 88: 834; Gould et al., (1989)
J. Cell Biol 108: 1657; Creissen et al., (1991) Plant J. 2: 129;
Kalderon et al., (1984) Cell 39: 499; Stiefel et al., (1990) Plant
Cell 2: 785).
B. Foreign Genes that may be Introduced into Tobacco Plants.
With transgenic plants according to the present invention, a
foreign protein can be produced in commercial quantities. Thus,
techniques for the selection and propagation of transformed plants,
which are well understood in the art, yield a plurality of
transgenic plants, which are harvested in a conventional manner,
and a foreign protein can then be extracted from a tissue of
interest or from total biomass. Protein extraction from plant
biomass can be accomplished by known methods which are discussed,
for example, by Heney and Orr, (1991) Anal. Biochem. 114: 92.
According to a preferred embodiment, the transgenic tobacco plant
is provided for commercial production of foreign protein. A genetic
map can be generated, for example, via conventional Restriction
Fragment Length Polymorphisms (RFLP), Polymerase Chain Reaction
(PCR) analysis, and Simple Sequence Repeats (SSR), which identifies
the approximate chromosomal location of the integrated DNA
molecule. For exemplary methodologies in this regard, see Glick and
Thompson, METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY
269-284 (CRC Press, Boca Raton, 1993). Map information concerning
chromosomal location is useful for proprietary protection of a
subject transgenic plant. If unauthorized propagation is undertaken
and crosses made with other germplasm, the map of the integration
region can be compared to similar maps for suspect plants, to
determine if the latter have a common parentage with the subject
plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR
and sequencing, all of which are conventional techniques.
Likewise, by means of the present invention, genes of agronomic
importance can be expressed in transformed plants. More
particularly, plants can be genetically engineered to express
various phenotypes of agronomic interest. Exemplary genes
implicated in this regard include, but are not limited to, those
described below.
For example, a trait transferred into cultivar NC 2000 may confer
resistance to brown stem rot (U.S. Pat. No. 5,689,035) or
resistance to cyst nematodes (U.S. Pat. No. 5,491,081). In a
preferred embodiment, a transgene whose expression results or
contributes to a desired trait to be transferred to cultivar NC
2000 comprises a gene encoding an insecticidal protein, such as,
for example, a crystal protein of Bacillus thuringiensis or a
vegetative insecticidal protein from Bacillus cereus, such as VIP3
(see, for example, Estruch et al. (1997) Nat Biotechnol 15:137). In
another preferred embodiment, a transgene introduced into cultivar
NC 2000 comprises a herbicide tolerance gene whose expression
renders plants of cultivar NC 2000 tolerant to the herbicide. For
example, expression of an altered acetohydroxyacid synthase (AHAS)
enzyme confers upon plants tolerance to various imidazolinone or
sulfonamide herbicides (U.S. Pat. No. 4,761,373). In another
preferred embodiment, a gene conferring tolerance to imidazolinones
or sulfonylurea herbicides is transferred into cultivar NC 2000.
Expression of a mutant acetolactate synthase (ALS) will render the
plants resistant to inhibition by sulfonylurea herbicides (U.S.
Pat. No. 5,013,659). In another preferred embodiment, U.S. Pat. No.
4,975,374, relates to plant cells and plants containing a gene
encoding a mutant glutamine synthetase (GS) resistant to inhibition
by herbicides that are known to inhibit GS, e.g., phosphinothricin
and methionine sulfoximine. In addition, expression of a
Streptomyces bar gene encoding a phosphinothricin acetyl
transferase results in tolerance to the herbicide phosphinothricin
or glufosinate (U.S. Pat. No. 5,489,520). U.S. Pat. No. 5,162,602
discloses plants tolerant to inhibition by cyclohexanedione and
aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred
by an altered acetyl coenzyme A carboxylase (ACCase). U.S. Pat. No.
5,554,798 discloses transgenic glyphosate tolerant plants, which
tolerance is conferred by an altered
5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene. In another
particular embodiment, tolerance to a protoporphyrinogen oxidase
inhibitor is achieved by expression of a tolerant
protoporphyrinogen oxidase enzyme in plants (U.S. Pat. No.
5,767,373). In another particular embodiment, a nucleic acid
transferred into cultivar NC 2000 comprises a transgene conferring
tolerance to a herbicide and at least one other transgene
conferring another trait, such as for example, insect resistance or
tolerance to another herbicide.
Other illustrative transgenes are set forth below.
1. Genes that Confer Resistance to Pests or Disease and that
Encode:
(A) Plant disease resistance genes. Plant defenses are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be
transformed with a cloned resistance gene to engineer plants that
are resistant to specific pathogen strains (see, for example, Jones
et al., (1994) Science 266: 789, cloning of the tomato Cf-9 gene
for resistance to Cladosporium fulvum; Martin et al., (1993)
Science 262: 1432, tomato Pto gene for resistance to Pseudomonas
syringae pv.; Mindrinos et al., (1994) Cell 78: 1089, Arabidopsis
RSP2 gene for resistance to Pseudomonas syringae).
(B) A Bacillus thuringiensis protein, a derivative thereof or a
synthetic polypeptide modeled thereon (see, for example, Geiser et
al., (1986) Gene 48: 109, disclosing the cloning and nucleotide
sequence of a Bt .delta.-endotoxin gene). Moreover, DNA molecules
encoding .delta.-endotoxin genes can be purchased from American
Type Culture Collection (Rockville, Md.), for example, under ATCC
Accession Nos. 40098, 67136, 31995 and 31998.
(C) A lectin (see, for example, the disclosure by Van Damme et al.,
(1994) Plant Molec. Biol. 24: 25), which discloses the nucleotide
sequences of several Clivia miniata mannose-binding lectin
genes.
(D) A vitamin-binding protein such as avidin (see PCT publication
WO 93/06487). This publication teaches the use of avidin and avidin
homologues as larvicides against insect pests.
(E) An enzyme inhibitor, for example, a protease inhibitor or an
amylase inhibitor (see, for example, Abe et al., (1987) J. Biol.
Chem. 262: 16793, nucleotide sequence of rice cysteine proteinase
inhibitor; Huub et al., (1993) Plant Molec. Biol. 21: 985;
nucleotide sequence of cDNA encoding tobacco proteinase inhibitor
1; and Sumitani et al., (1993) Biosci. Biotech. Biochem. 57: 1243,
nucleotide sequence of Streptomyces nitrosporeus amylase
inhibitor).
(F) An insect-specific hormone or pheromone such as an ecdysteroid
or juvenile hormone, a variant thereof, a mimetic based thereon, or
an antagonist or agonist thereof (see, for example, the disclosure
of Hammock et al., (1990) Nature 344: 458, of baculovirus
expression of cloned juvenile hormone esterase, an inactivator of
juvenile hormone).
(G) An insect-specific peptide or neuropeptide which, upon
expression, disrupts the physiology of the affected pest (for
example, see the disclosures of Regan, (1994) J. Biol. Chem. 269:
9, expression cloning yields DNA coding for insect diuretic hormone
receptor; Pratt et al., (1989) Biochem. Biophys. Res. Comm. 163:
1243, an allostatin is identified in Diploptera puntata). See also
U.S. Pat. No. 5,266,317 to Tomalski et al., which discloses genes
encoding insect-specific, paralytic neurotoxins.
(H) An insect-specific venom produced in nature by a snake, a wasp,
or the like (see, e.g., Pang et al., (1992) Gene 116: 165, for
disclosure of heterologous expression in plants of a gene coding
for a scorpion insectotoxic peptide).
(I) An enzyme responsible for an hyperaccumulation of a monterpene,
a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid
derivative or another non-protein molecule with insecticidal
activity.
(J) An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule;
for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic
enzyme, a nuclease, a cyclase, a transaminase, an esterase, a
hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase,
an elastase, a chitinase and a glucanase, whether natural or
synthetic (see PCT application WO 93/02197 in the name of Scott et
al., which discloses the nucleotide sequence of a callase gene).
DNA molecules which contain chitinase-encoding sequences can be
obtained, for example, from the ATCC under Accession Nos. 39637 and
67152 (see also Kramer et al., (1993) Insect Biochem. Molec. Biol.
23: 691, which describes the nucleotide sequence of a cDNA encoding
tobacco hookworm chitinase, and Kawalleck et al., (1993) Plant
Molec. Biol. 21: 673, which provides the nucleotide sequence of the
parsley ubi4-2 polyubiquitin gene).
(K) A molecule that stimulates signal transduction. For example,
see the disclosure by Botella et al., (1994) Plant Molec. Biol. 24:
757, of nucleotide sequences for mung bean calmodulin cDNA clones,
and Griess et al., (1994) Plant Physio. 104: 1467, which provides
the nucleotide sequence of a maize calmodulin cDNA clone.
(L) A hydrophobic moment peptide (see PCT application WO 95/16776
which disclosures peptide derivatives of Tachyplesin which inhibit
fungal plant pathogens, and PCT application WO 95/18855 which
teaches synthetic antimicrobial peptides that confer disease
resistance).
(M) A membrane permease, a channel former or a channel blocker. For
example, see the disclosure by Jaynes et al., (1993) Plant Sci. 89:
43), of heterologous expression of a cecropin-.beta.lytic peptide
analog to render transgenic tobacco plants resistant to Pseudomonas
solanacearum.
(N) A viral-invasive protein or a complex toxin derived therefrom.
For example, the accumulation of viral coat proteins in transformed
plant cells imparts resistance to viral infection and/or disease
development effected by the virus from which the coat protein gene
is derived, as well as by related viruses (see Beachy et al.,
(1990) Ann. Rev. Phytopathol. 28: 451). Coat protein-mediated
resistance has been conferred upon transformed plants against
alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus,
potato virus X, potato virus Y, tobacco etch virus, tobacco rattle
virus and tobacco mosaic virus (Id.).
(O) An insect-specific antibody or an immunotoxin derived
therefrom. Thus, an antibody targeted to a critical metabolic
function in the insect gut would inactivate an affected enzyme,
killing the insect (Cf. Taylor et al., Abstract #497, SEVENTH INT'L
SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh,
Scotland, 1994; enzymatic inactivation in transgenic tobacco via
production of single-chain antibody fragments).
(P) A virus-specific antibody (see, for example, Taviadoraki et
al., (1993) Nature 366: 469; showing that transgenic plants
expressing recombinant antibody genes are protected from virus
attack).
(Q) A developmental-arrestive protein produced in nature by a
pathogen or a parasite. Thus, fungal endo
.alpha.-1,4-D-polygalacturonases facilitate fungal colonization and
plant nutrient release by solubilizing plant cell wall
homo-.alpha.-1,4-D-galacturonase (see Lamb et al., (1992)
Bio/Technology 10: 1436). The cloning and characterization of a
gene which encodes a bean endopolygalacturonase-inhibiting protein
is described by Toubart et al., (1992) Plant J. 2: 367.
(R) A developmental-arrestive protein produced in nature by a
plant. For example, Logemann et al., (1992) Bio/Technology 10: 305,
have shown that transgenic plants expressing the barley
ribosome-inactivating gene have an increased resistance to fungal
disease.
2. Genes that Confer Resistance to a Herbicide, for Example:
(A) An herbicide that inhibits the growing point or meristem, such
as an imidazalinone or a sulfonylurea. Exemplary genes in this
category code for mutant ALS or AHAS enzyme as described, for
example, by Lee et al., (1988) EMBO J. 7: 1241, and Miki et al.,
(1990) Theor. Appl. Genet 80: 449, respectively.
(B) Glyphosate (resistance imparted by mutant
5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes) and
other phosphono compounds such as glufosinate (phosphinothricin
acetyl transferase (PAT) and Streptomyces hygroscopicus
phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or
phenoxy proprionic acids and cycloshexones (ACCase
inhibitor-encoding genes). See, for example, U.S. Pat. No.
4,940,835 to Shah et al., which discloses the nucleotide sequence
of a form of EPSP which can confer glyphosate resistance. A DNA
molecule encoding a mutant aroA gene can be obtained under ATCC
accession No. 39256, and the nucleotide sequence of the mutant gene
is disclosed in U.S. Pat. No. 4,769,061 to Comai. European patent
application No. 0 333 033 to Kumada et al. and U.S. Pat. No.
4,975,374 to Goodman et al. disclose nucleotide sequences of
glutamine synthetase genes which confer resistance to herbicides
such as L-phosphinothricin. The nucleotide sequence of a
phosphinothricin-acetyl-transferase gene is provided in European
application No. 0 242 246 to Leemans et al. De Greef et al., (1989)
Bio/Technology 7: 61, describe the production of transgenic plants
that express chimeric bar genes coding for phosphinothricin acetyl
transferase activity. Exemplary of genes conferring resistance to
phenoxy proprionic acids and cycloshexones, such as sethoxydim and
haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by
Marshall et al., (1992) Theor. Appl. Genet. 83: 435.
(C) An herbicide that inhibits photosynthesis, such as a triazine
(psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla
et al., (1991) Plant Cell 3: 169, describe the transformation of
Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide
sequences for nitrilase genes are disclosed in U.S. Pat. No.
4,810,648 to Stalker, and DNA molecules containing these genes are
available under ATCC Accession Nos. 53435, 67441 and 67442. Cloning
and expression of DNA coding for a glutathione S-transferase is
described by Hayes et al., (1992) Biochem. J. 285: 173.
3. Genes that Confer or Contribute to a Value-Added Trait, Such
as:
(A) Altered chemical composition, for example, an increase or
decrease in nicotine, total alkaloid, or reducing sugar
content.
(B) Decreased phytate content:
(i) Introduction of a phytase-encoding gene would enhance breakdown
of phytate, adding more free phosphate to the transformed plant.
For example, see Van Hartingsveldt et al., (1993) Gene 127: 87, for
a disclosure of the nucleotide sequence of an Aspergillus niger
phytase gene.
(C) Modified carbohydrate composition effected, for example, by
transforming plants with a gene coding for an enzyme that alters
the branching pattern of starch (see Shiroza et al., (1998) J.
Bacteriol. 170: 810, nucleotide sequence of Streptococcus mutans
fructosyltransferase gene; Steinmetz et al., (1985) Mol. Gen.
Genet. 200: 220, nucleotide sequence of Bacillus subtilis
levansucrase gene; Pen et al., (1992) Bio/Technology 10: 292,
production of transgenic plants that express Bacillus licheniformis
.alpha.-amylase; Elliot et al., (1993) Plant Molec. Biol. 21: 515,
nucleotide sequences of tomato invertase genes; S.o slashed.gaard
et al., (1993) J. Biol. Chem. 268: 22480, site-directed mutagenesis
of barley .alpha.-amylase gene; and Fisher et al., (1993) Plant
Physiol. 102: 1045, maize endosperm starch branching enzyme
II).
Those skilled in the art will appreciate that the transgenes
described above may also be transferred into tobacco plants using
conventional breeding techniques as known in the art and as
described herein.
As a further alternative, the transgene encodes an antisense RNA
molecule or any other non-translated RNA as known in the art. In a
further alternative embodiment, the transgene effects gene
suppression in the host plant.
C. Methods for Tobacco Transformation.
Plants can be transformed according to the present invention using
any suitable method known in the art. Intact plants, plant tissue,
explants, meristematic tissue, protoplasts, callus tissue, cultured
cells, and the like may be used for transformation depending on the
plant species and the method employed. In a preferred embodiment,
intact plants are inoculated using microprojectiles carrying a
nucleic acid to be transferred into the plant. The site of
inoculation will be apparent to one skilled in the art; leaf tissue
is one example of a suitable site of inoculation. In preferred
embodiments, intact plant tissues or plants are inoculated, without
the need for regeneration of plants (e.g., from callus).
Exemplary transformation methods include biological methods using
viruses and Agrobacterium, physicochemical methods such as
electroporation, polyethylene glycol, ballistic bombardment,
microinjection, and the like.
In one form of direct transformation, the vector is microinjected
directly into plant cells by use of micropipettes to mechanically
transfer the recombinant DNA (Crossway, Mol. Gen. Genetics 202: 179
(1985)).
In another protocol, the genetic material is transferred into the
plant cell using polyethylene glycol (Krens, et al. Nature 296, 72
(1982)).
In still another method, protoplasts are fused with minicells,
cells, lysosomes, or other fusible lipid-surfaced bodies that
contain the nucleotide sequence to be transferred to the plant
(Fraley, et al., Proc. Natl. Acad. Sci. USA 79, 1859 (1982)).
DNA may also be introduced into the plant cells by electroporation
(Fromm et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)). In this
technique, plant protoplasts are electroporated in the presence of
plasmids containing the expression cassette. Electrical impulses of
high field strength reversibly permeabilize biomembranes allowing
the introduction of the plasmids. Electroporated plant protoplasts
reform the cell wall, divide and regenerate. One advantage of
electroporation is that large pieces of DNA, including artificial
chromosomes, can be transformed by this method.
Viruses include RNA and DNA viruses (e.g., geminiviruses,
badnaviruses, nanoviruses and caulimoviruses).
Ballistic transformation typically comprises the steps of: (a)
providing a plant tissue as a target; (b) propelling a
microprojectile carrying the heterologous nucleotide sequence at
the plant tissue at a velocity sufficient to pierce the walls of
the cells within the tissue and to deposit the nucleotide sequence
within a cell of the tissue to thereby provide a transformed
tissue. In particular preferred embodiments of the invention, the
method further includes the step of culturing the transformed
tissue with a selection agent. In particular embodiments, the
selection step is followed by the step of regenerating transformed
plants from the transformed tissue. As noted below, the technique
may be carried out with the nucleotide sequence as a precipitate
(wet or freeze-dried) alone, in place of the aqueous solution
containing the nucleotide sequence.
Any ballistic cell transformation apparatus can be used in
practicing the present invention. Exemplary apparatus are disclosed
by Sandford et al. (Particulate Science and Technology 5, 27
(1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356.
Such apparatus have been used to transform maize cells (Klein et
al., Proc. Natl. Acad. Sci. USA 85, 4305 (1988)), soybean callus
(Christou et al., Plant Physiol. 87, 671 (1988)), McCabe et al.,
BioTechnology 6, 923 (1988), yeast mitochondria (Johnston et al.,
Science 240, 1538 (1988)), and Chlamydomonas chloroplasts (Boynton
et al., Science 240, 1534 (1988)).
Alternately, an apparatus configured as described by Klein et al.
(Nature 70, 327 (1987)) may be utilized. This apparatus comprises a
bombardment chamber, which is divided into two separate
compartments by an adjustable-height stopping plate. An
acceleration tube is mounted on top of the bombardment chamber. A
macroprojectile is propelled down the acceleration tube at the
stopping plate by a gunpowder charge. The stopping plate has a
borehole formed therein, which is smaller in diameter than the
microprojectile. The macroprojectile carries the
microprojectile(s), and the macroprojectile is aimed and fired at
the borehole. When the macroprojectile is stopped by the stopping
plate, the microprojectile(s) is propelled through the borehole.
The target tissue is positioned in the bombardment chamber so that
a microprojectile(s) propelled through the bore hole penetrates the
cell walls of the cells in the target tissue and deposit the
nucleotide sequence of interest carried thereon in the cells of the
target tissue. The bombardment chamber is partially evacuated prior
to use to prevent atmospheric drag from unduly slowing the
microprojectiles. The chamber is only partially evacuated so that
the target tissue is not desiccated during bombardment. A vacuum of
between about 400 to about 800 millimeters of mercury is
suitable.
In alternate embodiments, ballistic transformation is achieved
without use of microprojectiles. For example, an aqueous solution
containing the nucleotide sequence of interest as a precipitate may
be carried by the macroprojectile (e.g., by placing the aqueous
solution directly on the plate-contact end of the macroprojectile
without a microprojectile, where it is held by surface tension),
and the solution alone propelled at the plant tissue target (e.g.,
by propelling the macroprojectile down the acceleration tube in the
same manner as described above). Other approaches include placing
the nucleic acid precipitate itself ("wet" precipitate) or a
freeze-dried nucleotide precipitate directly on the plate-contact
end of the macroprojectile without a microprojectile. In the
absence of a microprojectile, it is believed that the nucleotide
sequence must either be propelled at the tissue target at a greater
velocity than that needed if carried by a microprojectile, or the
nucleotide sequenced caused to travel a shorter distance to the
target tissue (or both).
It is currently preferred to carry the nucleotide sequence on a
microprojectile. The microprojectile may be formed from any
material having sufficient density and cohesiveness to be propelled
through the cell wall, given the particle's velocity and the
distance the particle must travel. Non-limiting examples of
materials for making microprojectiles include metal, glass, silica,
ice, polyethylene, polypropylene, polycarbonate, and carbon
compounds (e.g., graphite, diamond). Metallic particles are
currently preferred. Non-limiting examples of suitable metals
include tungsten, gold, and iridium. The particles should be of a
size sufficiently small to avoid excessive disruption of the cells
they contact in the target tissue, and sufficiently large to
provide the inertia required to penetrate to the cell of interest
in the target tissue. Particles ranging in diameter from about
one-half micrometer to about three micrometers are suitable.
Particles need not be spherical, as surface irregularities on the
particles may enhance their DNA carrying capacity.
The nucleotide sequence may be immobilized on the particle by
precipitation. The precise precipitation parameters employed will
vary depending upon factors such as the particle acceleration
procedure employed, as is known in the art. The carrier particles
may optionally be coated with an encapsulating agents such as
polylysine to improve the stability of nucleotide sequences
immobilized thereon, as discussed in EP 0 270 356 (column 8).
Alternatively, plants may be transformed using Agrobacterium
tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium
tumefaciens. Agrobacterium-mediated gene transfer exploits the
natural ability of A. tumefaciens and A. rhizogenes to transfer DNA
into plant chromosomes. Agrobacterium is a plant pathogen that
transfers a set of genes encoded in a region called T-DNA of the Ti
and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively,
into plant cells. The typical result of transfer of the Ti plasmid
is a tumorous growth called a crown gall in which the T-DNA is
stably integrated into a host chromosome. Integration of the Ri
plasmid into the host chromosomal DNA results in a condition known
as "hairy root disease". The ability to cause disease in the host
plant can be avoided by deletion of the genes in the T-DNA without
loss of DNA transfer and integration. The DNA to be transferred is
attached to border sequences that define the end points of an
integrated T-DNA.
Gene transfer by means of engineered Agrobacterium strains has
become routine for many dicotyledonous plants. Some difficulty has
been experienced, however, in using Agrobacterium to transform
monocotyledonous plants, in particular, cereal plants. However,
Agrobacterium mediated transformation has been achieved in several
monocot species, including cereal species such as rye (de la Pena
et al., Nature 325, 274 (1987)), maize (Rhodes et al., Science 240,
204 (1988)), and rice (Shimamoto et al., Nature 338, 274
(1989)).
While the following discussion will focus on using A. tumefaciens
to achieve gene transfer in plants, those skilled in the art will
appreciate that this discussion also applies to A. rhizogenes.
Transformation using A. rhizogenes has developed analogously to
that of A. tumefaciens and has been successfully utilized to
transform, for example, alfalfa, Solanum nigrum L., and poplar.
U.S. Pat. No. 5,777,200 to Ryals et al. As described by U.S. Pat.
No. 5,773,693 to Burgess et al., it is preferable to use a disarmed
A. tumefaciens strain (as described below), however, the wild-type
A. rhizogenes may be employed. An illustrative strain of A.
rhizogenes is strain 15834.
The Agrobacterium strain is typically modified to contain the
nucleotide sequences to be transferred to the plant. The nucleotide
sequence to be transferred is incorporated into the T-region and is
typically flanked by at least one T-DNA border sequence, preferably
two T-DNA border sequences. A variety of Agrobacterium strains are
known in the art, and can be used in the methods of the invention.
See, e.g., Hooykaas, Plant Mol. Biol. 13, 327 (1989); Smith et al.,
Crop Science 35, 301 (1995); Chilton, Proc. Natl. Acad. Sci. USA
90, 3119 (1993); Mollony et al., Monograph Theor. Appl. Genet NY
19, 148 (1993); Ishida et al., Nature Biotechnol. 14, 745 (1996);
and Komari et al., The Plant Journal 10, 165 (1996).
In addition to the T-region, the Ti (or Ri) plasmid contains a vir
region. The vir region is important for efficient transformation,
and appears to be species-specific.
Two exemplary classes of recombinant Ti and Ri plasmid vector
systems are commonly used in the art. In one class, called
"cointegrate," the shuttle vector containing the gene of interest
is inserted by genetic recombination into a non-oncogenic Ti
plasmid that contains both the cis-acting and trans-acting elements
required for plant transformation as, for example, in the PMLJ1
shuttle vector of DeBlock et al., EMBO J. 3, 1681 (1984), and the
non-oncogenic Ti plasmid pGV2850 described by Zambryski et al.,
EMBO J. 2, 2143 (1983). In the second class or "binary" system, the
gene of interest is inserted into a shuttle vector containing the
cis-acting elements required for plant transformation. The other
necessary functions are provided in trans by the non-oncogenic Ti
plasmid as exemplified by the pBIN19 shuttle vector described by
Bevan, Nucleic Acids Research 12, 8711 (1984), and the
non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al.,
Nature 303, 179 (1983).
Binary vector systems have been developed where the manipulated
disarmed T-DNA carrying the heterologous nucleotide sequence of
interest and the vir functions are present on separate plasmids. In
this manner, a modified T-DNA region comprising foreign DNA (the
nucleic acid to be transferred) is constructed in a small plasmid
that replicates in E. coli. This plasmid is transferred
conjugatively in a tri-parental mating or via electroporation into
A. tumefaciens that contains a compatible plasmid with virulence
gene sequences. The vir functions are supplied in trans to transfer
the T-DNA into the plant genome. Such binary vectors are useful in
the practice of the present invention.
In particular embodiments of the invention, super-binary vectors
are employed. See, e.g., U.S. Pat. No. 5,591,615 and EP 0 604 662.
Such a super-binary vector has been constructed containing a DNA
region originating from the hypervirulence region of the Ti plasmid
pTiBo542 (Jin et al., J. Bacteriol. 169, 4417 (1987)) contained in
a super-virulent A. tumefaciens A281 exhibiting extremely high
transformation efficiency (Hood et al., Biotechnol. 2, 702 (1984);
Hood et al., J. Bacteriol. 168, 1283 (1986); Komari et al., J.
Bacteriol. 166, 88 (1986); Jin et al., J. Bacteriol. 169, 4417
(1987); Komari, Plant Science 60, 223 (1987); ATCC Accession No.
37394.
Exemplary super-binary vectors known to those skilled in the art
include pTOK162 (Japanese patent Appl. (Kokai) No. 4-222527, EP
504,869, EP 604,662, and U.S. Pat. No. 5,591,616) and pTOK233
(Komari, Plant Cell Reports 9, 303 (1990); Ishida et al., Nature
Biotechnology 14, 745 (1996)). Other super-binary vectors may be
constructed by the methods set forth in the above references.
Super-binary vector pTOK162 is capable of replication in both E.
coli and in A. tumefaciens. Additionally, the vector contains the
virB, virC and virG genes from the virulence region of pTiBo542.
The plasmid also contains an antibiotic resistance gene, a
selectable marker gene, and the nucleic acid of interest to be
transformed into the plant. The nucleic acid to be inserted into
the plant genome is typically located between the two border
sequences of the T region. Super-binary vectors can be constructed
having the features described above for pTOK162. The T-region of
the super-binary vectors and other vectors for use in the invention
are constructed to have restriction sites for the insertion of the
genes to be delivered. Alternatively, the DNA to be transformed can
be inserted in the T-DNA region of the vector by utilizing in vivo
homologous recombination. See, Herrera-Esterella et al., EMBO J. 2,
987 (1983); Horch et al., Science 223, 496 (1984). Such homologous
recombination relies on the fact that the super-binary vector has a
region homologous with a region of pBR322 or other similar
plasmids. Thus, when the two plasmids are brought together, a
desired gene is inserted into the super-binary vector by genetic
recombination via the homologous regions.
Plant cells may be transformed with Agrobacteria by any means known
in the art, e.g., by co-cultivation with cultured isolated
protoplasts, or transformation of intact cells or tissues. The
first requires an established culture system that allows for
culturing protoplasts and subsequent plant regeneration from
cultured protoplasts. Identification of transformed cells or plants
is generally accomplished by including a selectable marker in the
transforming vector, or by obtaining evidence of successful
bacterial infection.
In plants stably transformed by Agrobacteria-mediated
transformation, the nucleotide sequence of interest is incorporated
into the plant genome, typically flanked by at least one T-DNA
border sequence. Preferably, the nucleotide sequence of interest is
flanked by two T-DNA border sequences.
Plant cells, which have been transformed by any method known in the
art, can also be regenerated to produce intact plants using known
techniques.
Plant regeneration from cultured protoplasts is described in Evans
et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan
Publishing Co. New York, 1983); and Vasil I. R. (ed.), Cell Culture
and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I,
1984, and Vol. II, 1986). It is known that practically all plants
can be regenerated from cultured cells or tissues, including but
not limited to, all major species of sugar-cane, sugar beet,
cotton, fruit trees, and legumes.
Means for regeneration vary from species to species of plants, but
generally a suspension of transformed protoplasts or a petri plate
containing transformed explants is first provided. Callus tissue is
formed and shoots may be induced from callus and subsequently root.
Alternatively, somatic embryo formation can be induced in the
callus tissue. These somatic embryos germinate as natural embryos
to form plants. The culture media will generally contain various
amino acids and plant hormones, such as auxin and cytokinins. It is
also advantageous to add glutamic acid and proline to the medium,
especially for such species as corn and alfalfa. Efficient
regeneration will depend on the medium, on the genotype, and on the
history of the culture. If these three variables are controlled,
then regeneration is usually reproducible and repeatable.
A large number of plants have been shown capable of regeneration
from transformed individual cells to obtain transgenic whole
plants.
The regenerated plants are transferred to standard soil conditions
and cultivated in a conventional manner. The plants are grown and
harvested using conventional procedures.
The particular conditions for transformation, selection and
regeneration may be optimized by those of skill in the art. Factors
that affect the efficiency of transformation include the species of
plant, the tissue infected, composition of the media for tissue
culture, selectable marker genes, the length of any of the
above-described step, kinds of vectors, and light/dark conditions.
Therefore, these and other factors may be varied to determine what
is an optimal transformation protocol for any particular plant
species. It is recognized that not every species will react in the
same manner to the transformation conditions and may require a
slightly different modification of the protocols disclosed herein.
However, by altering each of the variables, an optimum protocol can
be derived for any plant species.
The foregoing methods for transformation may be used for producing
transgenic inbred or doubled-haploid lines. Transgenic
inbred/doubled-haploid lines could then be crossed, with another
(non-transformed or transformed) inbred or doubled-haploid line, in
order to produce a transgenic hybrid tobacco plant. Alternatively,
a genetic trait which has been engineered into a particular tobacco
line using the foregoing transformation techniques could be moved
into another line using traditional backcrossing techniques that
are well known in the plant breeding arts. For example, a
backcrossing approach could be used to move an engineered trait
from a non-elite line into an elite tobacco line, or from a hybrid
tobacco plant containing a foreign gene in its genome into a line
or lines which do not contain that gene. As used above, "crossing"
can refer to a simple X by Y cross, or the process of backcrossing,
depending on the context.
VI. Industrial Applicability
This invention is also directed to methods for producing a tobacco
plant by crossing a first parent tobacco plant with a second parent
tobacco plant wherein either the first or second parent tobacco
plant is a tobacco plant of cultivar NC 2000 or a tobacco plant of
cultivar NC 2000 further comprising one or more additional traits
(e.g., single gene traits). Further, both first and second parent
tobacco plants can come from cultivar NC 2000 or a tobacco plant of
cultivar NC 2000 further comprising one or more traits (e.g.,
single gene traits). Thus, any such methods using the tobacco
cultivar NC 2000 or a tobacco plant of NC 2000 further comprising
one or more additional traits (e.g., one or more single gene
traits) are part of this invention: selfing, backcrosses,
doubled-haploid production, hybrid production, crosses to
populations, and the like. All plants produced using tobacco
cultivar NC 2000 or modified cultivar NC 2000 further comprising
one or more additional traits (e.g., one or more single gene
traits) as a parent are within the scope of this invention.
Advantageously, tobacco cultivar NC 2000 or modified cultivar NC
2000 further comprising one or more additional traits (e.g., one or
more single gene traits) are used in crosses with other, different,
tobacco inbreds or doubled-haploids to produce first generation
(F.sub.1) tobacco hybrid seeds and plants with superior
characteristics.
As used herein, the term "plant" includes plant cells, plant
protoplasts and plant tissue cultures from which tobacco plants can
be regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants, such as leaves, pollen,
embryos, cotyledon, hypocotyl, roots, root tips, anthers, flowers
and parts thereof, ovules, shoots, stems, stalks, pith, capsules,
and the like.
As used herein, the term "tissue culture" encompasses cultures of
tobacco tissue, cells, protoplasts and callus. Methods of culturing
tobacco tissue, cells, protoplasts and callus, as well as methods
of regenerating plants from tobacco tissue cultures are described
in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen.
Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W.
H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761
pp. Thus, another aspect of this invention is to provide cells
which upon growth and differentiation produce tobacco plants having
the physiological and morphological characteristics of tobacco
cultivar NC 2000. In a preferred embodiment, cells of cultivar NC
2000 are transformed genetically, for example with one or more
genes described above, and transgenic plants of tobacco cultivar NC
2000 are regenerated therefrom.
VII. Deposits.
A deposit of at least 2500 seeds of tobacco cultivar NC 2000 has
been deposited with the American Type Culture Collection (ATCC),
Manassas, Va. 20110 USA on Sep. 21, 2001. The deposit has been
assigned ATCC Accession Number PTA-3721. This deposit of the
tobacco cultivar NC 2000 will be maintained in the ATCC depository,
which is a public depository, for a period of 30 years, or 5 years
after the most recent request, or for the effective life of the
patent, whichever is longer, and will be replaced if it becomes
nonviable during that period.
Having now described the invention, the same will be illustrated
with reference to certain examples, which are included herein for
illustration purposes only, and which are not intended to be
limiting of the invention.
EXAMPLE 1
To the inventor's knowledge, NC 2000 is most similar to one of its
parents, KY 14; however, NC 2000 is highly resistant to blue mold
caused by the fungal pathogen Peronospora tabacina Adam (see Table
3), whereas KY 14 is susceptible.
The NC BMR-113 parent also exhibits resistance to blue mold.
However, NC 2000 is a cultivar, whereas NC BMR-113 is a germplasm
line that is not adapted or suitable for commercial
cultivation.
Blue Mold Resistance:
Field evaluations of blue mold resistance in NC 2000 and check
cultivars were conducted at the Mountain Research Station in
Waynesville, N.C. and at the Upper Mountain Research Station in
Laurel Springs, N.C. in years 1995-1998. NC 2000 was entered into
the Regional Burley Variety Evaluation Test in 1998. In addition to
evaluations conducted in the burley growing belt, evaluations for
blue mold reaction were also performed under natural epidemics in
Papantla, Veracruz, Mexico.
Blue mold resistance in NC 2000 is manifested by reduced number and
size of lesions with minimum sporulation. Based on the studies at
the two North Carolina research stations, NC 2000 is resistant
against multiple isolates of the blue mold fungus, including
Metaxyl-sensitive and Metaxyl-resistant strains. Tables 1 through
4, below, were compiled from data obtained from the 1998 Regional
Burley Variety Evaluation Test, and from experiments conducted at
the Mountain and Upper Mountain Research Stations and Papantla,
Veracruz, Mexico from 1995-1998. Table 3 presents data related to
percent leaf area damaged due to blue mold for cultivars NC 2000,
KY 14, TN 86 and OVENS 62 (a flue-cured blue mold resistant
cultivar). A score was given to each plot according to the
percentage of the leaf area damaged (% LAD), and then that score
was transformed into a geometric mean (Horsfall and Barrett, (1945)
Phytopathology 36:655).
Other Characteristics:
Similar to KY 14, NC 2000 is also resistant to tobacco mosaic virus
(TMV) and wildfire (Pseudomonas syringae pv. tabaci), and is
susceptible to black shank (races 1 and 0), black root rot, and the
polyvirus complex (see Table 4). Leaf yield of NC 2000 is similar
to KY 14 (see, Tables 1 and 2).
Tobacco cultivar NC 2000 also differs from KY 14 in the following
respects:
Time to maturity for NC 2000 is approximately nine days later than
for KY 14.
NC 2000 is approximately 12 centimeters shorter than KY 14 (topped)
and approximately 13 centimeters shorter than KY 14 (not
topped).
The leaves of NC 2000 are approximately 1 to 9 cm shorter and 1 to
2.5 centimeters narrower than those of KY 14.
TABLE 1 Mean yield (lbs/acre), grade index and chemical composition
of check cultivars and NC 2000 in the 1998 Regional Small Plot
tests grown at seven locations. Yield Grade Nicotine Variety lbs./A
Index % KY 14 2623 69 4.10 VA 509 2638 72 4.73 NC 2000 2348 69
4.95
TABLE 2 Mean yield (lbs/acre), grade index and chemical composition
of check cultivars and NC 2000 grown at the Upper Mountain Research
Station (UMRS), Laurel Springs, NC and the Mountain Research
Station (MRS), Waynesville, NC in 1995, 1996, 1997, and 1998. UMRS
Yield MRS Yield Mean Yield Grade Total Variety lbs./A lbs./A lbs./A
Index Alkaloids 1995 KY 14 2785 2036 2411 71 3.78 TN 86 2944 2358
2651 71 3.32 NC 2000 2658 2282 2470 69 2.75 1996 KY 14 2105 1048
1577 68 2.54 TN 86 2205 1248 1727 70 2.31 NC 2000 2196 1114 1655 68
2.71 1997 KY 14 2127 1361 1744 65 3.58 TN 86 2207 1484 1846 68 2.87
NC 2000 2210 1155 1682 73 3.67 1998 KY 14 2005 2612 2309 57 3.63 VA
509 2362 2875 2619 70 3.28 TN 86 2184 2641 2413 64 2.85 NC 2000
2476 2766 2621 65 3.28
TABLE 3 Evaluation of Percent Leaf Area Damaged (% LAD) due to blue
mold (Peronospora tabacina Adam) at the Upper Mountain Research
Station (UMRS), Laurel Springs, NC, the Mountain Research Station
(MRS), Waynesville, NC and Papantla, Veracruz, Mexico in 1995,
1996, 1997, and 1998. UMRS MRS MEXICO 1995 KY 14 26.4 -- 98.6 TN 86
28.1 -- 91.9 OVENS 62 1.4 -- 9.1 NC 2000 12.7 -- 15.7 1996 KY 14
35.2 82.6 66.8 TN 86 19.2 28.1 67.2 OVENS 62 1.4 1.4 2.1 NC 2000
1.4 13.8 8.6 1997 KY 14 10.3 56.1 78.2 TN 86 5.9 9.4 15.1 OVENS 62
0.0 1.4 2.1 NC 2000 1.4 1.4 5.3 1998 KY 14 19.7 3.5 29.5 TN 86 8.5
3.3 19.9 OVENS 62 0.5 0.2 1.0 NC 2000 1.4 1.4 2.8 *Blue Mold
disease pressure was low in the mountain regions of North Carolina
in 1995 and 1998.
TABLE 4 Regional Small Plot burley tobacco disease ratings.sup.1,
1998. TOBACCO TOBACCO BLACK SHANK BLACK MOSAIC ETCH WILD VARIETY
Race 0 Race 1 ROOT ROT VIRUS VIRUS FIRE KY 14 S S MS R S R VA 509
MS R MS S S R NC 2000 S S S R S R .sup.1 Disease ratings reported
as R = resistant, S = susceptible, and MS = moderately
susceptible.
EXAMPLE 2
Materials and Method
Identification of Markers Linked to the BMR Locus
Traditional breeding methods are difficult to use when breeding for
blue mold resistance. Having to wait for natural epidemics to occur
increases the interval between cycles of selection. The interaction
between host and pathogen is extremely complex which causes disease
reactions to be highly variable, unpredictable, and often
non-reproducible. The use of molecular markers could reduce the
amount of time and effort required to identify resistance in burley
tobacco.
Population Development.
Two burley breeding lines released from the North Carolina
Agricultural Research Service in 1992, NCBMR-113 and NCBMR-114,
were used as the maternal parents and crossed with TN 90.
Maternally derived doubled haploid lines were obtained through the
N. africana method and chromosome doubled using the leaf midvein
technique.
Laboratory Screening.
Fifty newly developed doubled haploid (DH) lines were screened for
blue mold resistance using molecular markers found to be linked to
the target gene. NCBMR-113 and NCBMR-114 and OVENS 62 were used as
the resistant controls and TN 86, TN 90 and KY 14 were used as
susceptible controls.
DNA Extraction.
Seed from 50 maternally-derived doubled haploid lines were sown in
plastic pots on Metro-Mix 220.TM. (Milpitas, Calif.) growing
medium. Growing conditions were kept constant at 24.degree. C.
under a 16 hour day and 9 hour night regime for approximately 8
weeks. At the 7 to 10 leaf stage tissue was taken and ground for
twenty seconds with disposable pestles in 1.5 mL Eppendorf tubes.
Four hundred microliters of extraction buffer (PEC: 20 mM Tris HCl,
pH 7.5, 25 mM NaCl, 25 mM MEDTA, 0.5% SDS) was immediately added.
Tubes were vortexed to disperse tissue evenly in solution. Samples
were incubated for a minimum of ten minutes. DNA extracts were
centrifuged for 1 min at 13000 rpm and 300 .mu.l of the supernatant
was transferred to a new tube along with the addition of 300 .mu.l
of isopropanol. Samples were incubated at room temperature for 2
minutes and then centrifuged for 5 minutes at 13000 rpm.
Supernatant was discarded and 300 .mu.l of 70% ethanol was added.
Solution was centrifuged at 13000 rpm to form a DNA pellet, air
dried and resuspended in 100 .mu.l of TE buffer. DNA was
centrifuged for 2 minutes at 10000 rpm and supernatant transferred
to a new tube. Samples were stored at 4.degree. C.
RAPD Analysis.
PCR was carried out using 10-mer primers of arbitrary sequence on a
PTC-100.TM.MJ Research Programmable Thermal Controller (MJ
Research, Inc., Watertown, Mass.). Each 15 .mu.l of master mix
contained 4 .mu.l DNA 10.times.PCR buffer, 200 mM dNTPS (dATP,
dCTP, dGTP, dTTP), 1 unit Taq DNA Polymerase Stoffel fragment, 4 mM
MgCl.sub.2, 10% BSA and 20 ng primer. Gels were run in a Horizon
20-25 horizontal gel electrophoresis apparatus at 65V for a period
of six hours and then visualized on an UV transilluminator.
Field Screening.
The fifty lines were evaluated in Papantla, Veracruz, Mexico for
resistance to blue mold under natural conditions. Entries were
replicated three times in a randomized block design. Experimental
units consisted of one-row plots containing twelve plants per row.
Based on the Horsfall-Barrett disease rating scale a score was
given to each plot according to the percentage of the leaf area
damaged (% LAD) and then that score was transformed into a
geometric mean.
EXAMPLE 3
Results of Marker Analysis
Forty primers detected polymorphic bands between the susceptible
and resistant bulks, but only 21 produced bands that were linked to
the gene conditioning resistance to blue mold (BMR) after
individual DNA amplification of the lines comprising the bulks
(Table 5). Six of these primers (UBC-149, UBC-180, UBC-534,
UBC-544, UBC-610, UBC-240) were selected due to their repeatability
and ease of scoring to use in pre-screening (FIGS. 1 and 2).
Out of the fifty newly classified DH lines, twenty-nine were
classified as resistant when they were pre-screened using molecular
markers. Of these twenty-nine, only fifteen had % LAD of 10% of
less warranting a resistant classification in the field
evaluations. Twenty-one out of the fifty DH lines were classified
as susceptible using the markers. Of these twenty-nine lines,
twenty were classified as susceptible in the field (FIG. 3). It is
proposed that this one line that was not in agreement is a
recombinant. It showed the highest level of resistance of all lines
tested, including controls, in the field evaluation with a 2.3%
LAD. After conducting both field and laboratory evaluations of the
fifty previously unclassified doubled haploid lines it was found
that the agreement between field reaction and marker classification
was only 70% with reliability being higher at the extremes of
resistance and susceptibility to blue mold (FIG. 3).
The investigations described in this and the previous Example are
described in more detail in Milla et al. (Susana R. Milla,
Identification of RAPD Markers Linked to Blue Mold Resistance in
Tobacco, Master's Thesis, North Carolina State University,
1998).
TABLE 5 SEQ Type Size Quality LINES Sequence ID of of frag. of
SUSCEPTIBLE BULK PRIMER 5'- to -3' NO: marker (bp) amp..sup.a Ky 14
Ky 17 TN 86 TN 90 Speight G-28 Speight G-70 McNair 944 OPAE-02
TCGTTCACCC 1 coupling 335 *** 0.sup.b 0 0 0 0 0 0 OPAE-07
GTGTCAGTGG 2 repulsion 316 ** 1 1 1 1 1 1 1 OPAG-20 TGCGCTCCTC 3
coupling 416 ** 0 0 0 0 0 0 0 OPC-09 CTCACCGTCC 4 coupling 670 ** 0
0 0 0 0 0 0 OPP-11 AACGCGTCGG 5 coupling 663 *** 0 0 0 0 0 0 0
OPR-06 GTCTACCGCA 6 coupling 268 * 0 0 0 0 0 0 0 UBC-024 ACAGGGGTGA
7 coupling 589 ** 0 0 0 0 0 0 0 UBC-149 AGCAGCGTGG 8 coupling 228
*** 0 0 0 0 0 0 0 UBC-180 GGGCCACGCT 9 coupling 328 ** 0 0 0 0 0 0
0 UBC-240 ATGTTCCAGG 10 repulsion 545 ** 1 1 1 1 1 1 1 UBC-243
GGGTGAACCG 11 repulsion 335 *** 1 1 1 1 1 1 1 UBC-528 GGATCTATGC 12
coupling 528 *** 0 0 0 0 0 0 0 UBC-534 CACCCCCTGC 13 coupling 436
*** 0 0 0 0 0 0 0 UBC-544 TAGAGACTCC 14 coupling 499 ** 0 0 0 0 0 0
0 UBC-563 CGCCGCTCCT 15 coupling 566 ** 0 0 0 0 0 0 0 UBC-610
TTTGCCGCCC 16 coupling 528 ** 0 0 0 0 0 0 0 UBC-624 GTGATAAGCC 17
coupling 480 ** 0 0 0 0 0 0 0 LINES RESISTANT BULK PRIMER Ovens 62
DH 17 DH 62 NC-BMR-42 NC-BMR-90 NC-BMR-113 NC-BMR-114 OPAE-02 1 1 0
1 1 1 1 OPAE-07 0 0 1 0 0 0 0 OPAG-20 1 1 0 1 1 1 1 OPC-09 1 1 0 1
1 1 1 OPP-11 1 1 0 1 1 1 1 OPR-06 1 1 0 1 1 1 1 UBC-024 1 1 0 1 1 1
1 UBC-149 1 1 0 1 1 1 1 UBC-180 1 1 0 1 1 1 1 UBC-240 0 0 1 0 0 0 0
UBC-243 0 0 1 0 0 0 0 UBC-528 1 1 0 1 1 1 1 UBC-534 1 1 0 1 1 1 1
UBC-544 1 1 0 1 1 1 1 UBC-563 1 1 0 1 1 1 1 UBC-610 1 1 0 1 1 1 1
UBC-624 1 1 0 1 1 1 1 .sup.a * = fair, ** = good, *** = very good
.sup.b 0 = absence of the marker, 1 = presence of the marker
Having now described the invention, the same will be illustrated
with reference to certain examples, which are included herein for
illustration purposes only, and which are not intended to be
limiting of the invention.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 17 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 1 tcgttcaccc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Repulsion marker <400>
SEQUENCE: 2 gtgtcagtgg 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 3 tgcgctcctc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 4 ctcaccgtcc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 5 aacgcgtcgg 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 6 gtctacggca 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 7 acaggggtga 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 8 agcagcgtgg 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 9 gggccacgct 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Repulsion marker <400>
SEQUENCE: 10 atgttccagg 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Repulsion marker <400>
SEQUENCE: 11 gggtgaaccg 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 12 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 12 ggatctatgc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 13 caccccctgc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 14 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 14 tagagactcc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 15 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 15 cgccgctcct 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 16 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 16 tttgccgccc 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Coupling marker <400>
SEQUENCE: 17 gtgataagcc 10
* * * * *